1. Field of the Invention
The present invention relates to a biochip comprising a bio-receptor chemically or physicochemically attached to a high density carbon nanotube (CNT) film or pattern having chemical functional groups exposed, in which the bio-receptor is capable of binding to a target biomaterial, and a method for preparing the same.
2. Background of the Related Art
Carbon nanotube (CNT) is an allotrope of carbon, which consists of carbons exists abundantly on the earth. They are tubular materials where a carbon atom is connected to other carbons in the form of a hexagonal honeycomb structure. Their diameter is about size of nanometer ( 1/109 meter). CNT is known to have excellent mechanical properties, electrical selectivity, field emission properties and highly efficient hydrogen storage properties and be new and almost defect-free of all the existing materials.
Because of their properties of excellent structural rigidity, chemical stability, ability to act as ideal one-dimensional (1D) “quantum wires” with either semiconducting or metallic behaviors and a large aspect ratio, CNT exhibits a broad range of potential applications as a basic material of flat panel displays, transistors, energy reservoirs, etc., and as various sensors with nanosize (Dai, H., Acc. Chem. Res., 35:1035-1044, 2002).
In order to apply such properties more diversely, the purified single-walled CNT has been cut into short nanotube pieces using an acid. The cut CNT pieces have mainly —COOH chemical functional groups at a part of ends and sidewall of the open tube. The properties of the CNT have been modified by chemical binding of various materials using these chemical functional groups. Further, there have been reported that the functional group of CNT was substituted for —SH group by chemical manipulation and patterned on a gold surface (Nan, X. et al., J. Colloid Interface Sci., 245:311-8, 2002) and that CNT was immobilized on substrate using the electrostatic method (Rouse, J. H. et al., Nano Lett., 3:59-62, 2003). However, the former has disadvantages of the low CNT surface density and the weak bonding, and the latter also has a fatal disadvantage that the patterning method for selective immobilization on the surface cannot be applied. Therefore, there is an urgent demand for developing a new type surface immobilizing method with high density.
Recently, researches are being conducted to detect reactions both protein-protein and protein-ligand by means of electrochemical changes of CNT after immobilization of a biomaterial (Dai, H. et al., ACC. Chem. Res., 35:1035-44, 2002; Sotiropoulou, S. et al., Anal. Bioanal. Chem., 375:103-5, 2003; Erlanger, B. F. et al., Nano Lett., 1:465-7, 2001; Azamian, B. R. et al., JACS, 124:12664-5, 2002). A representative example of a protein-ligand reaction is an avidin-biotin reaction. Star et al. formed a channel on a substrate, which had been treated with a polymer, using CNT and measured the binding activity of streptoavidin by means of an electrochemical method (Star, A. et al., Nano Lett., 3:459-63, 2003).
The reasons that CNT attracts public attention as a biochip are as followings: Firstly, it needs no labeling; secondly, it has high sensitivity to signal change; and thirdly, it is capable of reacting in an aqueous solution without deterioration of a protein. The combination of a new nanomaterial and a biological system will create important fusion technologies in respective fields of disease diagnosis (hereditary diseases), proteomics and nanobiotechnology.
In order to develop a rapider and cheaper biochip, many researches have been conducted on technologies of DNA hybridization detection. Various labeling technologies for detecting DNA hybridization have been developed. An effective surface treatment capable of increasing hybridization efficiency and simultaneously, removing the background from non-specific binding is required to detect the DNA hybridization effectively using the DNA chip. Many researches have been conducted to prepare a surface-treated DNA chip platform (Rogers, Y. et al., Anal. Biochem., 266:23-30, 1999; Hu, J. et al., Nuc. Acid. Res., 29:106-10, 2001). Also, various methods for detecting DNA hybridization were developed, which include the scanometric method, the calorimetric method, a method using nanoparticle, a method using electrochemistry, and etc. (Taton, T. A. et al., Science, 289:1757-60, 2000; Alexandre, I. et al., Anal. Biochem., 295:1-8, 2001; Cai, H. et al., Analyst., 127:803-8, 2002; Cai, H. et al., Anal. Bioanal. Chem., 375:287-93, 2003).
Besides many applications with CNT in the bioengineering field have been recently being appeared. It is being suggested that the applicability of CNT to biochips, such as glucose biosensors, detecting protein, detecting a certain DNA sequence and the like (Sotiropoulou, S. et al., Anal. Bioanal. Chem., 375:103-5, 2003; Chen, R. J. et al., Proc. Natl. Acad. Sci. USA, 100:4984-9, 2003; Cai, H. et al., Anal. Bioanal. Chem., 375:287-93, 2003). Screening bio-molecules from multilayer based on CNT can increase the amount of immobilized bio-substances, such as DNAs and detecting sensitivity to the bio-substances, since the CNT has wide surface area and high electric conductivity.
At the present time, the most universal method for detecting the result of the reaction in a biochip is to use conventional fluorescent materials and isotopes (Toriba, A. et al., Biomed. Chromatogr., 17:126-32, 2003; Syrzycka, M. et al., Anal. Chim. Acta, 484:1-14, 2003; Grow, A. E. et al., J. Microbio. Meth., 53:221-33, 2003). However, as novel methods to easily and precisely measure an electrical or electrochemical signal are attempted, there are increased demands for CNT as a new material.
The methods comprising preparing a high density CNT multiplayer, attaching DNA thereon and detecting complementary DNA are useful in genotyping, mutation detection, pathogen identification and the like. It has been reported that PNA (peptide nucleic acid: DNA mimic) is regio-specifically fixed on a single walled CNT and the complementary binding to probe DNA is detected (Williams, K. A. et al., Nature, 420:761, 2001). Also, there has been an example, in which an oligonucleotide was fixed on a CNT array by a electrochemical method and DNA was detected by guanine oxidation (Li, J. et al., Nano Lett., 3:597-602, 2003). However, these methods do not apply CNT to fabrication and development of biochips.
Recently, a high capacity biomolecule detection sensor using CNT was disclosed (WO 03/016901 A1). This patent relates to a multi-channel type biochip produced by arranging a plurality of CNTs on a substrate using a chemical linker and attaching various types of receptors. However, it has a disadvantage of relative weakness to environmental changes.
Therefore, the present inventors have found a method for producing a CNT-biochip by repeated laminating CNT on a substrate having exposed amine groups by chemical bonding to form a high density CNT film or pattern having exposed chemical functional groups and chemically binding a bio-receptor to the CNT film or pattern, or treating the CNT surface with a chemical to prevent the non-specific binding by physical adsorption and chemically binding the bio-receptor to the treated surface, and completed the present invention.